System and Method for Pixelated Fluid Assay

ABSTRACT

A method of performing a fluid-material assay employing a device including at least one active pixel having a sensor with an assay site functionalized for selected fluid-assay material. The method includes exposing the pixel&#39;s sensor assay site to such material, and in conjunction with such exposing, and employing the active nature of the pixel, remotely requesting from the pixel&#39;s sensor assay site an assay-result output report. The method further includes, in relation to the employing step, creating, relative to the sensor&#39;s assay site in the at least one pixel, a predetermined, pixel-specific electromagnetic field environment.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Continuation of an application entitled,MICRO-PIXELATED ACTIVE-MATRIX FLUID-ASSAY PERFORMANCE, invented by JohnHartzell et al., Ser. No. 11/888,491, filed Jul. 31, 2007, attorneydocket no. SLA2242.5, which is incorporated by reference. Thisapplication claims filing-date priority to currently co-pending U.S.Provisional Patent Application Ser. No. 60/849,875, filed Oct. 6, 2006,for “Micro-Pixelated Array Assay Structure and Methodology”. The entiredisclosure content of that prior-filed provisional case is herebyincorporated herein by reference. The following applications, all filedon Jul. 10, 2007, are also incorporated herein by reference: Ser. No.11/827,174, Ser. No. 11/827,176, Ser. No. 11/827,175, Ser. No.11/827,335, and Ser. No. 11/827,173.

BACKGROUND AND SUMMARY OF THE INVENTION

This invention relates to the field of fluid-material assays. Moreparticularly, it relates to the performance of such an assay in thespecific context of employing a significantly improved type ofthin-film-based, active-pixel, pixelated assay-response matrix of thekind illustrated and described both in the above-mentioned provisionalapplication, and also in currently co-pending U.S. patent applicationSer. No. 11/827,174, filed Jul. 10, 2007, for “Micro-PixelatedFluid-Assay Structure”, the full disclosure content of which is herebyincorporated herein by reference.

This active-pixel matrix, which is a digitally accessible andcontrollable structure linkable to a suitable digital computer, offers avery high degree of controlled, assay-response, pixel-specificsensitivity with respect to which an assay response (a) can beoutput-read on a precision, pixel-by-pixel basis, and (b) canadditionally be examined along uniquely accessible, special, plural andfreely selectable, independent-variable “information-gathering axes”,such as a time-based axis, and an electromagnetic-field-variable (light,heat, non-uniform electrical) axis.

This matrix structure with its included electronically active pixels,which structure is preferably employed in the assay-performance practiceof the present invention, is

formed conveniently on a low-temperature substrate material, such asglass, and may involve, in its underlying construction, low-temperature,internal crystalline-structural processing of a material, such asamorphous silicon, to create some of its pixel-borne structuralfeatures. Such crystalline-structural processing is described in U.S.Pat. No. 7,125,451 B2, the disclosure content of which patent is alsohereby incorporated herein by reference.

More will be stated below herein regarding the interesting features ofthis representative matrix structure which make it so convenientlyuseable in the practice of the present invention.

So as to describe fully the important practice aspects of the presentmethod invention, those practice aspects are illustrated and discussedherein in relation to a specific form of pixelated matrix device—theform particularly set forth in depth in the '174 patent application. Itshould be understood, and it will become apparent, that other deviceforms may be employed, so long as these other forms include and displaycertain important structural and behavioral features principally focusedon the possession of what are referred to herein as individually,digitally computer addressable active pixels, or the like.

In general terms, the present invention may be described as a method ofperforming a fluid-material assay employing an appropriately provided(i.e., made available) computer-accessible device (note the discussionabove)—preferably a pixelated matrix device, including at least oneactive digitally-addressable-pixel having a sensor with adigitally-addressable assay site functionalized for selected fluid-assaymaterial, with the key steps of this method including, following, ofcourse, providing such a device, exposing the pixel's sensor assay siteto such material, and in conjunction with such exposing, and employingthe computer-accessible, active nature of the provided device's pixel,remotely and digitally requesting from the pixel's sensor assay site anassay-result output report.

The basic methodology further includes, in relation to the mentionedemploying step, creating, relative to the sensor's assay site in the atleast one pixel, a predetermined, pixel-specific electromagnetic fieldenvironment. The creation of such an environment is enabled by the typeof matrix structure described both hereinbelow, and in the '174 patentapplication, and is specifically enabled by the presence in thedescribed matrix pixels of one or several digitally accessible andenergizable electromagnetic field-creating structure(s).

The provided device, referred to above in the just-given generaldescription of the invention, may take on a number of different forms,not necessarily exactly the same as the device form specifically chosenherein to illustrate practice of the invention. Those skilled in the artwill appreciate from the disclosure of the invention provided in thisdocument, including the content in the mentioned currently pending,companion Regular U.S. patent application, just how to characterize apixelated device, and its relevant features and advantages, which willbe suitable for use during the performance of an assay in accordancewith implementation of the methodology of the present invention.

Accordingly, the various features and advantages of the herein proposedinvention methodology will become more fully apparent as the descriptionof the setting and a typical practice of the invention are presentedbelow in conjunction with the accompanying drawings.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a simplified, fragmentary, block/schematic view of a portionof a digitally-addressable, pixelated, fluid-assay, active-matrixmicro-structure—a device—which, when appropriately provided as part of apractice of the invention, is useable in relation to the preferred andbest mode manner of practicing the present invention.

FIG. 2 is similar to FIG. 1, except that it provides a slightly moredetailed view of the illustratively provided micro-structure shown inFIG. 1.

FIG. 3, which has been prepared on a somewhat larger scale than thosescales employed in FIGS. 1 and 2, illustrates, schematically, different,single-matrix/device organizational ways in which fluid-assay pixels ina matrix micro-structure like that shown in FIGS. 1 and 2 may bepre-organized into different functionalized arrangements for assistingdifferently in different fluid-assays that are to be performed inaccordance with practice of the present invention.

FIG. 4 is a fragmentary, block/schematic diagram showing apixel-specific, digitally accessible, electromagnetic field-creatingstructure which may be employed, in the practice of the presentinvention, in a device like that shown in FIGS. 1 and 2, andspecifically such a field-creating structure which is intended, underdigitally accessible computer control, to create a pixel-specificambient electromagnetic field environment characterized by light.

FIG. 5, which is functionally herein somewhat similar to FIG. 4,illustrates, fragmentarily, a pixel-specific, digitally accessibleheat-field-creating (electromagnetic field-creating) structure that hasbeen prepared on the body of a mechanical cantilever beam which alsocarries an electrical signaling structure that responds to beamdeflection during an assay performance to produce a related electrical,assay-result output signal. The specific structure of this figure isutilized later herein to discuss an illustrative DNA fluid assay whereinthe “provided device”, a matrix device, employsassay-site-functionalized (i.e., oligonucleotide-functionalized)micro-cantilever beams, such as that shown in this figure, to respond toDNA assay fluid under environmental conditions involving the creation ofpixel-specific heat fields (fixed and varying), and time-basedoutput-result sampling.

FIG. 6, which, categorically, is somewhat like FIGS. 4 and 5, provides asimplified side elevation of a pixel-specific, digitally accessible,non-uniform electrical-field-creating (electromagnetic field-creating)structure provided in a matrix micro-structure such as that pictured inFIGS. 1 and 2.

FIGS. 7-11, inclusive, provide block-schematic diagrams that illustratedifferent ways of viewing the methodologic practice steps of the presentinvention.

FIGS. 12-16, inclusive, help to describe various aspects, of theabove-mentioned, illustrative DNA fluid assay, with respect to which acontrolled heat field may be employed, and also time sampling may beused, to furnish different axes of assay-result output informationobtainable from practice of the present invention. This DNA-assayillustration provides a good basis for understanding the versatileutility of the invention with respect to both biologic and otherspecific types of fluid assays where electromagnetic-field axes, andtime axes, of output information may similarly be obtained.

DETAILED DESCRIPTION OF THE INVENTION Definitions

There is certain special terminology which is employed in thedescription and characterization of this invention—the meanings of whichterminology are presented immediately hereinbelow.

The terms “active-matrix” and “active pixel” as used herein refer to apixelated structure wherein each pixel is controlled by and in relationto some form of included, digitally-addressable electronic structure,which structure includes digitally-addressable electronic switchingstructure, defined by one or more electronic switching device(s),operatively associated both with also-included pixel-specificassay-sensor structure, and with pixel-bathing electromagneticfield-creating structure. Specifically, the “active” nature of an“active pixel” permits both (a) an assay-result output-report query tobe posed to the pixel, and (b) accessing and energizing of thereferred-to, associated pixel-bathing electromagnetic field-creatingstructure.

The term “bi-alternate” refers to an assay matrix structural conditionwherein every other pixel in each row and column of pixels is commonlyfunctionalized to possess response-affinity for one, specific type of afluid-material assay. This condition effectively creates, across theentire area of such an overall matrix, two differently functionalizedsubmatrices of pixels (what can be thought of as a two-assay,single-matrix condition).

The term “tri-alternate” refers to a similar condition, but one whereinevery third pixel in each row and column is commonly functionalized forone specific type of a fluid-material assay. This condition effectivelycreates, across the entire area of such an overall matrix, threedifferently functionalized submatrices of pixels (what can be thought ofas a three-assay, single-matrix condition).

Bi-alternate and tri-alternate matrices are discussed briefly in thisdisclosure to aid in understanding the depth of capability of the assayperformance methodology of the invention.

THE DRAWINGS

Turning attention now to the drawings, we set the stage for describingthe performance methodology of the present invention by describing,first, features of a “pre-assay-specific-performance”, to-be-provided,computer-digitally-accessible, pixelated assay matrix structure—a devicehaving functionalized pixels some of whose general characteristics andbehaviors, as will be explained, are relevant to practice of theinvention. Fundamentally, the invention is intended to be implemented inthe context of using a pixelated matrix device, or the like, formed withactive pixels which include assay sensors possessingassay-specific-functionalized assay sites, at least one each per pixel,that are individually, remotely, digitally addressable during theperformance of an assay to obtain assay-result output readings. In animportant sense, practice of the invention can take place even with anappropriate device having one such pixel.

Additionally, practice of the invention more specifically contemplatesthe presence, within each pixel of the type described above, of aremotely, individually digitally accessible and energizableelectromagnetic field-creating structure which, during an assay, may beenergized to create either a static (singular, stable) or atime-variable (staged, time variant) field condition useful variously toaffect/effect associated assay-sensor responses to exposure to an assayfluid. Appropriate fields include a field of light, a field of heat, anda non-uniform electrical (or electrical potential) field.

What now immediately follows is a description of a matrix structurewhich meets these assay-performance-support considerations—a structure,or device, which, in accordance with certain ways of describing thepresent invention, is provided as a lead step for practicing theinvention.

Beginning with FIGS. 1 and 2, indicated generally at 20 is a fragmentaryportion of a digitally-addressable, pixelated, fluid-assay,active-matrix micro-structure which takes the form herein of acolumn-and-row array 22 of plural, individually, externally,digitally-addressable pixels, such as those shown at 24, 26, 28, 30, 32,formed on an appropriate supporting substrate 34 made, for example, ofglass or plastic. For the purpose of illustration herein, substrate 34will be considered to be a glass substrate.

Various known fabrication practices, not relevant to the presentinvention, may be utilized to create the overall structure illustratedin FIGS. 1 and 2. More information about these practices will be foundin the text of the above-referred-to Ser. No. 11/827,174 patentapplication. Additionally, and with respect to matrix pixel structurestill to be discussed herein in relation to FIGS. 5 and 6, certainportions of that structure may conveniently be formed employing aninternal crystalline-structure processing approach such as thatdescribed in previously mentioned U.S. Pat. No. 7,125,451 B2.

While in no way critical to practice of the assay performancemethodology of the present invention, a conveniently useable pixelatedmicro-structure device, such as micro-structure 20, might have lateraldimensions lying in a range of about 0.4×0.4-inches to about 2×2-inches,and might include an equal row-and-column array of pixels including atotal pixel count lying in a range of about 100 to about 10,000.

Continuing with a description of what is shown in FIGS. 1 and 2, andconsidering an important communication pathway that is establishedduring practice of the present invention during an assay between apixelated device, such as matrix micro-structure 20, and a remotedigital computer, a bracket 36 and a double-headed, broad arrow 38 (seeFIG. 1) represent an appropriate communication/addressing connection, orpath, between pixels in micro-structure 20 and such a digital computer,which is shown in block form in FIG. 1 at 40.

Regarding the illustrated operative presence of a digital computer likecomputer 40, it should be understood that such a computer, while “remoteand external” with respect to the internal structures of the matrixpixels, per se, might actually be formed directly on-board a matrixsubstrate, such as substrate 34, or might truly be external to thissubstrate. In this context, it should be clearly understood thatcomputer location is not any part of the present invention.

In the particular construction of micro-structure 20 which isillustrated in FIGS. 1 and 2, each of the mentioned pixels isessentially identical to each other pixel, although, as will later beexplained herein, this might be different for the handling of differentspecific assays. This “might be different” statement is based upon anunderstanding, which should be made clear here, that there are variousfluid-assay applications with respect to which appropriatelyfunctionally differentiated pixels might be useful in an employed,micro-structure matrix array. Some of these differentiated-pixelconcepts, and how they fit with the practice methodology of the presentinvention, will be discussed more fully later herein.

In general terms, and using pixel 24 as an illustration to explain thebasic construction of each of the pixels shown in array 22, included inpixel 24 are several, fully integrated, pixel-specific components,subcomponents, or substructures. These include, as part of more broadlyinclusive pixel-specific electronic structure, (1) thin-film,digitally-addressable electronic switching structure, (2) a fullyassay-functionalized, individually remotely digitally-addressable andaccessible assay sensor 24 a which hosts a functionalized assay site 24a ₁, and (3) what is referred to herein as a pixel-bathing, ambientenvironmental, thin-film electromagnetic-field-creating structure 24 b.Field-creating structure 24 b, which is also remotely, or externally,individually digitally-addressable, accessible and energizable, isconstructed to create, when energized, any one or more of threedifferent kinds of pixel-bathing, assay-site-bathing, ambient,environmental electromagnetic fields in the vicinity of sensor 24 a.These three different field types include a light field, a heat field,and a non-uniform electrical field.

While structure 24 b, as was just mentioned, may be constructed tocreate one or more of these three different kinds of fields, in themicro-structure pictured in. FIGS. 1 and 2, field-creating structure 24b has been designed with three field-creating subcomponents 24 b ₁, 24 b₂ and 24 b ₃. Any one or more of these subcomponents may be computerenergized to create, within pixel 24, associated assay-site bathing,ambient field, conditions (“fixed-stable” or “time-variant”).Subcomponent 24 b ₁ is capable of creating a pixel-bathing light field,subcomponent 24 b ₂ a pixel-bathing heat field, and subcomponent 24 b ₃a pixel-bathing non-uniform electrical field.

Also included in pixel 24 is an optical detector 24 c. This detector,which is individually digitally addressable and accessible, may be usedto “read” any fluorescence output reaction generated by assay site 24 a,if and when that site is illuminated by light-field subcomponent 24 bduring the performance of a relevant assay, such as a DNA assay.

With regard to the active-matrix nature of micro-structure 20 which hasbeen chosen to illustrate practice of the present invention, it will beunderstood that each pixel is appropriately prepared with one or moreelectronic switching device(s) (part of the earlier-mentioned electronicswitching structure) relevant to accessing and addressing its includedsensor and assay site, and accessing and energizing its includedfield-creating structure. Digital addressing of, the electronicswitching structure in a pixel is also referred to herein as “employingthe active nature of the pixel”.

Looking specifically at FIG. 2 in relation to a further discussionregarding how a device, such as micro-structure 20, may be connected toa fully external computer (like computer 40) during the performance ofan assay in accordance with this invention, indicated generally at 42,44 are two different communication line systems which are operativelyconnected, respectively, to the field-creating structures in theillustrated pixels, and to the assay sensors and assay sites shown inthese pixels. The upper, fragmented ends of line systems 42, 44 in FIG.2 are embraced by a bracket marked 36, 38, which bracket represents thepreviously mentioned “path” of operative connection shown to exist inFIG. 1 between micro-structure 20 and computer 40. Line system 42, undercontrol of computer 40, is employable by this computer to access,digitally and individually, and to energize, pixel field-creatingsubcomponents during the performance of an assay procedure, andespecially, as will be explained, during requesting and obtaining areading-out, or outputting, of the results of a performed assay. Linesystem 44 directly and digitally couples, on a pixel-by-pixel,individual basis, to computer 40 assay-result output responses requestedand derived (obtained) from “assay-reacted” assay sites, and fromoptical detector 24 c. In order to avoid drawing clutter in FIG. 2, aspecific line extension from line 44 to detector 24 c has been omittedfrom this figure, although such an extension should be understood toexist.

Practice of the present invention in relation to the performance of anassay, as has been expressed already herein, involves, among otherthings, using an appropriately provided device having functionalizedassay sites, or at least one such site, which is (are) individuallydigitally addressable at least for the purpose of computer-requesting,and obtaining, assay-site reaction-output information. Preferably, sucha device includes a suitable plurality of functionalized assay sites,organized on, or in suitable relation to, an individually-digitallyaddressable and accessible assay sensor which forms part of anindividually digitally addressable and accessible pixel.

Preferably also, such a device's pixels additionally include one or moreelectromagnetic field-creating component(s), or subcomponents, whichis/are remotely, digitally accessible and energizable to create, on apixel-specific basis, a sensor-and-assay-site-bathing field, or fields,of light, heat and/or non-uniform electrical potential.

A matrix micro-structure, such as micro-structure 20, having pixelsdesigned with these characteristics, including one-each field-creatingsubcomponents covering each of the three different mentioned fields, isideal for this purpose, and thus has been chosen for use in illustratingand describing the practice of the present invention.

While the invention may very conveniently be practiced by aplural-pixelated device wherein all pixels are identicallyfunctionalized for a specific assay material, various kinds ofdifferent, functionalized-pixel-distribution patterns in a pixelatedmatrix device may be useful in certain assay-performance applications.FIG. 3 is provided herein to help illustrate this idea.

This figure illustrates several different such ways in whichcompleted-matrix, fully functionalized pixels, such as the pixels inarray 22, may be initially organized, and even differentiated, prior toprovision/delivery of a matrix to a user for use in the practice of thepresent invention. To begin with, and using the construction of matrixmicro-structure 20 specifically for illustration purposes the obvious,large dots, which appear throughout in a row-and-column arrangement inFIG. 3, represent the locations of next-adjacent pixels, such as thepixels in assay 22. One way of visualizing this matrix of pixels is toview the pixel arrangement as being one wherein every pixel representedby the mentioned dots is commonly functionalized to respond to, asingle, specific fluid-assay material.

Regions A, B, C in FIG. 3 illustrate three other, representative,possible pixel functionalization patterns (submatrix patterns) useablein the practice of the present invention.

In region A, which is but a small, or partial, region, or patch, of theoverall matrix array 22 of pixels, a functionalized submatrix patternexists, as illustrated by solid, horizontal and vertical intersectinglines, such as 48, 50, respectively, including rows and columns ofnext-adjacent pixels, which pixels are all commonly functionalized for aparticular fluid-material assay. With this kind of an arrangement,different patches, or fragmentary areas (i.e., unified lower-pixel-countsubmatrices defined by side-by-side pixels), of next-adjacent pixels maybe differently functionalized so that a single matrix array can be usedwith these kinds of patch submatrices to perform in plural, different,fluid-material assays.

In region B, intersecting, solid, horizontal and vertical lines, such aslines 52, 54, respectively, and intersecting, dashed, horizontal andvertical lines, such as lines 56, 58, respectively, illustrate two,different submatrix functionalization patterns which fit each into thecategory mentioned earlier herein as a bi-alternate functionalizationpattern which effectively creates two, large-area-distributionsubmatrices within the overall matrix array 22 of pixels. These twopixel submatrices are distributed across the entire area of the overallmatrix array, and are characterized by rows and columns of pixels which“sit” two pixel spacings away from one another.

Fig. C illustrates another submatrix functionalization pattern whereinintersecting, light; solid, horizontal and vertical lines, such as lines60, 62, respectively, intersecting dashed, horizontal and verticallines, such as lines 64, 66, respectively, and intersecting, thickened,solid, horizontal and vertical lines, such as lines 68, 70,respectively, represent what was referred to herein earlier as atri-alternate functionalization arrangement distributed over the entirematrix array 22 of pixels—effectively dividing that array into threeoverlapping submatrices.

Those skilled in the art, looking at the illustrative, suggestedfunctionalization patterns illustrated in FIG. 3, will understand howthese, and perhaps other, functionalization patterns will interestinglytap the utility of the assay-performance methodology of the presentinvention.

Continuing with an introductory description of matrix micro-structure 20which has been selected to illustrate a pixelated device employable inthe practice of the invention, and turning attention now to FIG. 4, thisfigure illustrates, schematically and fragmentarily, one style of aconventionally structured light-field-creating subcomponent. Thissubcomponent, with respect to what has been shown and discussed earlierherein regarding FIGS. 1 and 2, might sit at the field-creatingsubcomponent location which is labeled 24 b ₁ in FIGS. 1 and 2.

Thus, shown specifically in FIG. 4 is an energizable, optical medium 72which is computer-energized/switched directly by the operation of acontrol transistor (an electronic switching device) shown in block format 74. This control transistor has an operative connection to previouslymentioned line system 42. A sinuous arrow 76 extends from medium 72toward prospective assay site 24 a, which is hosted on sensor 24 a.Arrow 76 schematically pictures the creation of a field of light in thevicinity of site 24 a ₁. Light from medium 72 is characterized by anysuitable pre-chosen wavelength, and may be output from the medium, undercomputer control, at different controllable intensities (i.e., differentfield intensities).

Directing attention now to FIG. 5, here there is illustrated,schematically, an electronically (computer) switchable and intensity(temperature)-variable heat-field-creating subcomponent, which, while itmay be disposed at the location generally designated 24 b ₂ in FIG. 1,herein is deployed somewhat differently, and specifically, convenientlyat the location of an on-sensor-24 a site 88 which is formed directly onthe beam 90 a of a cantilever-type micro-deflection device 90 whose bodyhas been formed utilizing the process referred to above as internalcrystalline-structure processing (see U.S. Pat. No. 7,235,451 B2).

Also formed on beam 90 a is an electrical signaling structure 92 whichmay take the form of any suitable electrical device that responds tobending in beam 90 a to produce a related electrical output signal whichmay be coupled from the relevant pixel ultimately to an externalcomputer, such as computer 40.

Directing attention now to FIG. 6, this figure illustrates aspects of anelectronically (computer) switchable and intensity-variable,non-uniform-electrical-field-creating structure 94 which may be createdwithin a pixel, such as within pixel 24 at the site shown at 24 b ₃ inFIGS. 1 and 2. The mechanical spike structures seen in this figure havebeen fabricated employing the crystalline-structure-processingmethodology described in the above-referred-to '451 B2 U.S. patent.

Turning attention now to FIGS. 7-11, inclusive, and recognizing thatassay performance in accordance with practice of the present inventionis based upon use of a suitably provided, i.e., made-available, devicelike micro-structure 20, these five drawing figures illustrate the basichigh-level methodology of the invention which is practiceable inconjunction with such a device.

Speaking about the invention methodology, in the simplest terms, adevice, like micro-structure 20 with appropriately functionalizedpixels, sensors and assay sites is provided for use, and is placed in anassay-fluid environment, such as within a conventional flow-cell. Acomputer, like computer 40, is appropriately linked to the sensors,assay sites and field-creating structures in the device's pixels viacommunication/addressing path structure 36, 38, and the device's pixelsare then appropriately exposed to assay-fluid in the assay environment.

In conjunction with such exposure, and typically, though notnecessarily, beginning at the start of this exposure, under the controlof the relevant computer, one-by-one the pixels are digitallyaddressed/accessed to request from their respective sensors and assaysites assay-reaction output results/information so as to obtain, collectand store if desired, and report on, that information.

This pixel-by-pixel digital addressing may also be accompanied veryeffectively by simultaneous accessing and energizing of pixel-specificfield-creating subcomponents to produce one or more kind(s) of field(s),such as light, heat and electrical potential (or electrical gradient)fields, in the vicinities of addressed sensor assay sites in order toenhance assay-result information output. For example, with respect to agiven pixel assay site, output readings may be acquired at different,computer-controlled, static, or varying, electromagnetic fieldconditions, such as varying field-intensity conditions, and this mayalso be done in a sampling fashion on a time base, thus to openopportunities for gaining multiple “axes” of assay-result outputinformation.

With this general practice description in mind, FIG. 7, which includesthree blocks 100, 102, 104, illustrates one specific way of visualizingthe practice of the invention. From this point of view, the inventioncan be expressed as being a method of performing a fluid-material assayutilizing a device including at least one active pixel having a sensorwith an assay site functionalized for selected fluid-assay material,including the steps, following providing of the mentioned device, of (a)exposing the pixel's sensor assay site to such material (block 100), andin conjunction with such exposing, and (b) employing the active natureof the pixel (block 102), (c) remotely requesting from the pixel'ssensor assay site an assay-result output report (block 104).

Adding, to what is shown in FIG. 7, the presence of block 106 shown inFIG. 8, the method pictured effectively in FIG. 7, and just expressedabove, can be viewed as further including, in relation to the employing(block 104) step, the included, or related, or linked, step of creating,relative to the mentioned sensor's assay site (in the at least onepixel) a predetermined electromagnetic field environment (block 106).

FIG. 9 shows, in four blocks 108, 110, 112, 114, several other ways ofvisualizing the practice of the assay performance methodology of thepresent invention. Looking at this figure from a point of view whichfocuses on blocks 108, 110, 112, the invention can be expressed as beinga method for performing a fluid-material assay utilizing a pixelatedassay matrix wherein each pixel possesses an assay sensor with afunctionalized assay site, and is individually and remotely digitallyaddressable via the presence in the pixel of an active, selectivelyenergizable electronic switching structure which is operativelyconnected to the sensor and its assay site. The method steps from thisviewpoint include, following providing of mentioned matrix device, (a)subjecting the matrix to an environment containing assay fluid in orderto effect pixel-sensor assay-site reactions (block 108), in connectionwith this subjecting step, (b) remotely, digitally and individuallyaddressing selected pixel's included electronic switching structure(block 110), and (c), by that addressing step, requesting from thesensors' assay sites in the addressed pixels pixel-specific assay-resultoutput information (block 112).

Adding block 114 into the method statement described with regard toblocks 108, 110, 112, this additional block (114) illustrates theadditional step, which is a consequence of the requesting step, ofobtaining from each of the selected pixels' sensors' assay sties aresult-output reading of any reaction associated with that pixel'sincluded assay-sensor assay site.

FIG. 10 in the drawings illustrates, at least partially by blocks 110,116, 118, a further description of the invention methodology which isbased upon use of an assay support device wherein each pixel furtherincludes individually remotely and digitally accessible and energizableelectromagnetic field-creating structure that is both associated withthe pixel's assay sensor, and also operatively connected to the pixel'sincluded electronic switching structure. This figure describes themethodology, as expressed above in relation to FIG. 9 in an augmentedfashion by stating that the addressing step (block 110) further includesremotely, digitally and individually accessing and energizing a selectedpixel's field-creating structure (block 116), and by that accessing andenergizing step, creating, with respect to each selected pixel, apredetermined, pixel-specific electromagnetic field environment whichexists within that pixel in operative proximity to the pixel'sassociated assay sensor and its associated assay site (block 118).

FIG. 11 illustrates with a block 120 that, from an additionalperspective the just-described “creating” step includes the step ofproviding at least one of (a) a singular, stable, and (b) a staged,time-variant, electromagnetic field environment of the type generallymentioned in relation to the description of FIG. 10. It is also the casethat this producing (block 120) step includes the selectable practice ofproviding different pixel-specific electromagnetic field environmentswith respect to different pixels.

Turning attention now to FIGS. 12-16, inclusive, and to discussionrelevant thereto, these six figures are provided to illustrate issues,and resolutions thereof, involving a specific type of fluid-materialassay which is performable in accordance with practice of the presentinvention. Recognizing that the methodology of the invention may be usedwith a wide variety of different fluid-material assays, thisparticular-assay-type illustration will serve, in conjunction with thedescription of the invention which has been given so far herein, toinform those generally skilled in the art about the versatile utility ofthe present invention.

Very specifically, the illustration now to be described relates to theperformance of a DNA fluid-material assay utilizing a matrix constructedin accordance with the above-described features of micro-structure 20,and with the pixels in this micro-structure more specificallyconstructed in accordance with a sensor structure of the cantileverstyle which is illustrated in FIG. 5 in the drawings.

Even more specifically, the description of this illustrative practice ofthe invention will be given in the context of utilizing acomputer-controlled, environmental heat field employed during theperformance of an assay to enrich the obtainability of usefulassay-result output information from the illustrative DNA assay. Thedescription given now with respect to utilizing a heat-field device withrespect to creating an ambient electromagnetic field in the vicinity offunctionalized assay sites, should illustrate, successfully to thoseskilled in the art, how such a field, or others of the three differenttypes of electromagnetic fields referred to herein, utilized eithersingly or in different combinations, may help to offer significantlyimproved output information with respect to the conducting offluid-material assays.

Additionally, the illustration which now follows respecting a DNA-typeassay will be described, at least in part, in the context of varying aheat-field condition during the performance of an assay, andadditionally, in the context of taking time-spaced readings, as, forexample, by a sampling technique, to include, in addition to aheat-field axis of assay-result output information, also a time-basedaxis of such information.

The area of, and tasks involved with, DNA assays, the issues that thisassay field has raised in conventional practice, and the strikinglysuccessful moves toward resolutions of those issues offered by thepresent invention, dictate why we choose this DNA assay field for acertain amount of illustrative focused discussion herein. These DNA“issues”, and our invention's moves toward addressing them resolutelywill serve well to convey how the practice of the present invention isshaped to deal especially and innovatively with other fluid-assay areas.Accordingly, the specific DNA assay description which now follows stepsbriefly into the conventional background of the performance of DNAassays, and does so in a manner, and in a context, which compares thenovel utility and versatility of the present invention with prior artDNA assay practice.

In broad-brush terms, a DNA assay, aspects of which are now to bediscussed, is performed utilizing a provided, pixelated matrix includingappropriately functionalized sensors possessing predetermined (and notnecessarily all the same) oligonucleotide probes. This matrix is placedin a suitable fluid-assay environment, such as within a conventionalflow-cell, and fluid-assay material is introduced into that environment.A computer which is suitably connected operatively to the matrix'sactive pixels is employed, as desired, to request assay-result outputinformation on a pixel-by-pixel basis, and also to access and energizethe associated, pixel-specific heat-field-creating structures on atime-stable or time-varying basis to add interesting and highlyinformative output information.

We now progress a somewhat more detailed discussion regarding DNA assaysthrough five topical zones, A-E, inclusive, identified by relevant sideheadings below. The texts relating to these “zones” variously blend theissues of the “past” with resolutions of the “now” offered by thepresent invention.

A language-term family of DNA art which appears in thesetexts—hybridize, hybridized and hybridization—refers to theassay-important act of affinity bonding which occurs during assayperformance between a functionalized assay-site (an oligonucleotideprobe in the DNA world) and an assay-fluid component (a DNA or RNAmolecule, referred to as a target).

A. Functionalized Assay Site

Currently, and here discussing conventional heating-based DNA assays,all oligonucleotide probes employed in DNA-assay arrays can only behybridized at substantially the same temperature, since each such wholearray is heated simultaneously during assay performance. Thus, to obtainoptimum array performance in the past, it has been essential to designoligonucleotide probes with melting temperatures that lie within arelatively narrow melt-temperature window, usually of about 5° C. Thisrequirement for substantially uniform probe melting temperatures reducesassay flexibility, and puts serious constraints on probe-designalgorithms.

Individual heating devices positioned in close vicinity to probes, suchas the heat-field-creating structures proposed herein for use in thepractice of our present invention, will enable one to hybridize eachprobe at a different temperature. Thus, instead of initially preparing,within an array of probes, probes with melting temperatures that liewithin narrow temperature windows, it becomes possible to utilizeindividual hybridization temperatures matched to melting temperaturesfor different probes.

Recognizing that practice of the present invention contemplates, inpart, pre-assay-per-se-performance provision of a device prepared inadvance to have suitably functionalized assay sites associated withpixel-specific assay sensors, one intending to perform a particular DNAassay with such differently melt-temperature functionalized probes mayreadily specify an appropriate matrix functionalization “pattern”designed to accommodate this requirement. While a matrix supplier maychoose different ways to meet such a request, one very effective way fordoing so is described in U.S. patent application Ser. No. 11/827,173filed on Jul. 10, 2007 for “Micro-Pixelated Fluid-Assay Structure WithOn-Board, Addressable, Pixel-Specific Functionalization”. The fulldisclosure content of that patent application is hereby incorporatedherein by reference.

This option of using melt-temperature differentiation is verysignificant for some DNA-assay applications where, for example, probesare designed for really short sequences, and there is no easyopportunity for probe selection. A typical example of such anapplication involves one aimed at the detection of micro-RNA expressionimportant for cancer research.

B. Assay Confidence

A major issue relating to conventional DNA-assay arrays is so-calledbackground signal associated with non-specific binding of labeledtargets. Such binding can be caused by cross-hybridization of targetswith similar heterologous probes, and by random non-specific attachmentof targets to probes distributed over a matrix array surface.Cross-hybridization to heterologous probes depends on hybridizationtemperature, and can be decreased by precise temperature adjustment inthe vicinity of probes. In addition, non-specific binding differs fromspecific target-probe hybridization in terms of temperature dependence,and these two processes can be clearly distinguished by utilizing the“additional information axis” capability of the present invention,thereby obtaining a temperature-to-binding dependence category of outputinformation. A detected binding signal that does not match a profile forthe specific, intended interaction can be considered to be a falsepositive signal.

The ability, thus, to perform hybridization of a target DNA or RNAmolecule with multiple identical probes at different temperatures, as isreadily accommodated by the present invention, allows one tocharacterize the temperature dependence of target hybridization. Thisdependence can be used as a fingerprint approach for specifictarget-probe interactions, and it can be used to discriminate falsepositive signals on a matrix array. For so-called SNP (Single NucleotidePolymorphism) assays, those skilled in the relevant art will appreciatethat this approach will result in robust distinguishing between mutantand wild-type alleles.

C. SNP Assay

SNP discovery and detection is a very important area of DNA assayapplications in basic research and clinical diagnostics. The ability todistinguish the so-called wild-type DNA target molecule from one thathas a single sequence mismatch is based on different target-to-probebinding behaviors at different temperatures. For example, FIG. 12 showsthe typical expected temperature dependence of target-to-probe bindingfor a wild-type DNA (0wt), and for three, corresponding mismatches 0At,0Ac, and 0Ag.

This theoretical plot demonstrates that a clear difference in bindingfor so-called wild-types and mismatches can only be obtained in arelatively broad, rather than in a very narrow, temperature range. If awhole matrix array of probes (of functionalized assay sites) is heatedsimultaneously, it will thus, most probably be quite difficult toachieve optimum distinction for all probes. Each set of probes mayrequire a certain temperature range that is different from thoserequired by other sets of probes.

Generally, the temperature-dependence profiles of target-probehybridizations, if acquired in accordance with the practice of thepresent invention here different individual probes essentially“function” at different predetermined temperatures, can readily be readto distinguish not only between wild-type oligonucleotides andmismatches, but also between mismatches. FIG. 13, which illustratesthis, shows representative temperature-to-binding-dependence curves, orplots, that would be obtained typically by using an array of numerousoligonucleotide assay probes for such a set of targets where differentprobes in this array are designed for, and are hybridized at, differenttemperatures. Temperature variation in this setting will typically beperformed independently for groups of assay sites (probes) that havebeen commonly functionalized to possess replicates of the same probe.Thus FIG. 13 indicates that the measurement (and plotting) oftemperature-to-binding dependence will permit discrimination between thewild type and mismatching sequences as well as among differentmismatches.

Assay-site-specific sets of heating elements, as proposed for use incertain pixelated assay devices provided in conjunction with practice ofthe present invention, will contribute to a way to performhybridizations at different temperatures for individual probes withinone pixelated matrix array, and will result in accommodating theobtaining of temperature-to-binding profiles, like those pictured inFIG. 13, in a single test assay.

D. Target-to-Probe Hybridization at Time-Varying TemperaturesAccompanied with Real Time, Time-Axis Detection

Real-time, time-axis detection that is performed by a sensor and itsfunctionalized assay site at time-varying hybridization temperaturesallows one to obtain a unique individual pattern for eachtarget-to-probe interaction. FIG. 14A shows a typical, expectedhybridization signal for two different target and probe pairs at aconstant temperature. Saturation of the signal, illustrated in thisfigure, corresponds to the stage where hybridization equilibrium isachieved. If the two, pictured target-probe pairs I and II have aclosely similar sequence (for example, in the case of an SNP assay), theplots obtained for pairs I and II are difficult to distinguish. If,however, hybridization is performed at time-varying temperatures (FIG.14B), the resultant signal-to-time dependence plots have morecomplicated and perceivably different patterns. Such temperaturevariations (field-intensity variations/variants) are contemplated, ofcourse, as a useful possibility in the practice of the presentinvention.

At low temperatures, target-to-probe hybridization will cause anincrease in a detected binding signal. When the hybridizationtemperature exceeds the melting point for the subject target-probe pair,hybrids start to denature, causing a corresponding decrease in signal(see generally the right-side portion of FIG. 13). Thus, real-timedetection of hybridization signals at time-varying temperatures canprovide unique and readily distinguishable individual characteristicsfor each target-probe pair. For example, the upper “turn points” ofplots I and II in FIG. 14B can be used to distinguish highly similartarget sequences.

Temperature time varying can also be performed independently for severalsensing elements (assay sites) that contain (have been functionalized tocontain) replicates of the same probe. In such a case, the rate oftemperature increase (ΔT/Δtime) is different for different replicates.Thus, several signal-to-time dependence plots can be obtained for aparticular target-probe pair (see FIG. 15). These plots form a virtualthree-dimensional surface that is a fingerprint characteristic of ananalyzed target-probe pair.

These temperature time-varying illustrations not only describe herein atemperature-axis method for performing a DNA assay, they also illustratethat characteristic of the present invention which enables the obtainingof assay-result output information on a time-based axis, as by samplingon such an axis.

E. Active Thermal Oscillation of a Cantilever Transducer

Commonly, where a cantilever-style sensor is employed, a relevantcantilever “transducer signal” is associated with detection of acantilever deflection that is caused by a surface-tension change due tobio-interactions occurring on the cantilever surface at the location ofa functionalized assay site. The ability, offered during practice of thepresent invention, to vary, over time, the temperature in the cantilevervicinity allows for generation of a changing cantilever deflection.Thus, a “temperature oscillation” (see the light-colored, lower solidline in FIG. 16 in relation to the temperature-level axis which appearson the right side of this figure) results in a related, basicoscillation of cantilever response (see the darker, upper solid line inFIG. 16 in relation to the signal-level axis which appears on the leftside of this figure). Binding of bio-molecules to such a cantileversurface at the location of a functionalized assay site changes thesignal-axis pattern of the cantilever oscillation (see the dashed linein FIG. 16). Thus, such an oscillation pattern change can be used forquantification of bio-molecules which are assay-site-captured during anassay.

From the above-discussion regarding the performing of a representativeDNA assay wherein pixel-specific electromagnetic-field heat plays arole, and from the invention description given herein, it will beevident to those skilled in the art how other performance approaches maybe employed for conducting a DNA assay. For example, instead of using acantilever-type sensor in a device provided for the purpose ofperforming such an assay, one could alternatively employ a device havingpixels featuring non-cantilever, functionalized assay sites, andoffering the use of a pixel specific light field, and pixel-specificoptical detection, to query an assay-site for reaction-outputinformation during a DNA assay. Above-mentioned patent application Ser.No. 11/827,173 fully describes this kind of DNA-assay approach.

Further evident to those skilled in the art will be the fact that morethan a single type of electromagnetic field may be employed in thepractice of a DNA assay. For example, combined fields of light and heat,or other plural-combined fields, may be utilized.

The DNA-specific-assay discussion just presented above will also armthose skilled in the art with a clear understanding of how variousnon-DNA fluid-material assays may be conducted using the methodologyproposed by the present invention.

Accordingly, a preferred and best mode manner of practicing the presentinvention, and several modifications thereof, have been illustrated anddescribed herein. From these disclosures, those skilled in the relevantart will appreciate the numerous advances and advantages which areoffered by the invention in relation to the carrying out of variousdifferent types of fluid-material assays. Also, those so skilled willadditionally appreciate that other, currently unidentified variationsand modifications may come to their minds, and may be included in thepractice of the invention, with these variations and modifications beingclearly implementable without departing from the spirit of the inventionas set forth herein in the several claims to invention.

1-17. (canceled)
 18. An active matrix assay device comprising: aplurality of pixels formed on a substrate surface, each pixelcomprising: a digital communication interface; a digitally addressableelectronic switching structure connected to the communication interface;an assay site; and, a field-creation structure connected to theelectronic switching structure, the field-creation structure configuredto bathe the corresponding assay site with an energy field in responseto digital commands.
 19. The active matrix assay device of claim 18wherein the assay site is functionalized with a substance having anaffinity for an assay-specific-material.
 20. The active matrix assaydevice of claim 19 wherein each pixel further comprises: a sensorconnected to the electronic switching structure, the sensor configuredto detect assay-specific-material interactions with the functionalizedassay site, in response to digital commands.
 21. The active matrix assaydevice of claim 20 wherein the sensor is selected from a groupconsisting of an optical detector, and a strain detector to detecttarget molecule-induced deflections in an assay site formed on athin-film cantilever structure.
 22. The active matrix assay device ofclaim 20 wherein the assay site comprises an oligonucleotide probeattached to an assay site surface.
 23. The active matrix assay device ofclaim 22 wherein the sensor is configured to detect target moleculeinteractions with the oligonucleotide probe.
 24. The active matrix assaydevice of claim 18 wherein the field-creation structure is selected froma group consisting of a light-creating structure, a heat-creatingstructure, and a non-uniform electrical field-creating structure. 25.The active matrix assay device of claim 19 wherein a first plurality ofpixel assay sites are independently functionalized with substances tohave an affinity with a corresponding first plurality of differentassay-specific-materials.
 26. The active matrix assay device of claim 18wherein the substrate comprises a plurality of thin-film semiconductorlayers overlying a support substrate selected from a group consisting ofglass and plastic; and, wherein the assay site, field-creationstructure, and sensor are formed in the thin-film semiconductor layers.27. A method for functionalizing an active matrix assay device, themethod comprising: providing a device with a plurality of pixels formedon a substrate surface, each pixel comprising a digital communicationinterface, a digitally addressable electronic switching structure, anassay site, and a field-creation structure; addressing a first pixelfrom the plurality of pixels; selectively engaging the field-creationstructure in the first pixel; bathing the corresponding assay site withan energy field; and, in response to the energy field, functionalizingthe assay site with an affinity substance having an affinity for anassay-specific-material.
 28. The method of claim 27 further comprising:exposing the active matrix assay device to the affinity substance; and,wherein functionalizing the assay site includes functionalizing theassay site in response to the combination of the energy field and theaffinity substance exposure.
 29. The method of claim 28 wherein exposingthe active matrix assay device to the affinity substance includesexposing the active matrix assay device to an oligonucleotide; and,wherein functionalizing the assay site includes functiona the assay sitewith an oligonucleotide probe having an affinity for a particular DNAtarget molecule.
 30. The method of claim 28 wherein exposing the activematrix assay device to the affinity substance includes exposing theactive matrix assay device to a fluid including the affinity substance.31. The method of claim 27 wherein bathing the corresponding assay sitewith the energy field includes bathing with an energy field selectedfrom a group consisting of a light, heat, and a non-uniform electricalfield.
 32. The method of claim 27 wherein functionalizing the assay siteincludes independently functionalizing a first plurality of pixel assaysites with substances having an affinity with a corresponding firstplurality of different assay-specific-materials.
 33. A method forreading an active matrix assay device, the method comprising: providinga device with a plurality of pixels formed on a substrate surface, eachpixel comprising a digital communication interface, a digitallyaddressable electronic switching structure, a functionalized assay site,and a sensor; addressing a first pixel from the plurality of pixels;selectively engaging the sensor in the first pixel; and, in response toengaging the sensor, identifying an assay-specific-material interactingwith an affinity substance on the corresponding assay site.
 34. Themethod of claim 33 wherein providing the device functionalized assaysite includes providing a device where a plurality of pixels comprise anoligonucleotide probe attached to the assay site; and, whereinidentifying the assay-specific-material includes identifying a targetmolecule interacting with a corresponding assay site oligonucleotideprobe.
 35. The method of claim 34 wherein the sensor is an opticaldetector.
 36. The method of claim 33 wherein the assay site is formed ona thin-film cantilever; and, wherein the sensor is a strain detector todetect assay-specific-material-induced deflections in the thin-filmcantilever.
 37. The method of claim 33 wherein identifying theassay-specific-material includes independently identifying a firstplurality of assay-specific-materials corresponding with a firstplurality of different pixel assay sites.